Influenza, commonly known as “the flu”, is a respiratory infectious disease caused by the influenza virus, and has the symptoms of high fever, feeling tired, and muscle pains, accompanied by some respiratory symptoms. Influenza virus is a threat to human health, the sustained and rapid antigenic drift cause wide spread of seasonal influenza. According to statistics of WHO, seasonal influenza is responsible for the death of at least 250,000-500,000 persons per year (Peter D. C. et al., J Clin Invest. 2008, 118:3273-3275). In addition, flu outbreak is still an important threat to human beings. Since influenza virus was discovered, flu outbreak occurred five times around world in human history, resulting in tens of millions of deaths; among them, Spanish influenza (H1N1) in 1918 resulted in about 20-50 millions of deaths in the world. The influenza pandemics occurred in the 20th century also include Asian influenza (H2N2) in 1957 and Hong Kong Influenza (H3N2) in 1968, both of which caused serious public health threat and human social panic (Xu R. et al., Science. 2010, 328: 357-360). In the 21st century, influenza outbreaks still do not stop; influenza A (2009 pandemic H1N1) outbreak occurred in Mexico in 2009 and quickly spreaded around the world, which sounded the alarm to human society once again; according to WHO statistics, up to Aug. 6, 2010, a total of 18,449 cases of confirmed deaths were reported in more than 200 countries and regions globally (WHO Pandemic (H1N1) 2009—update 112. 6 Aug., 2010). In addition, human beings also face the threat of highly pathogenic avian influenza, and highly pathogenic H5N1 avian influenza virus has become one of the greatest infectious diseases that threats human beings, after “SARS” in 2003. Since 2003, 600 cases of human infected by avian influenza virus H5N1 are reported globally, among which 353 cases died, with a mortality rate close to 60% (WHO: http://www.who.int/influenza/human_animal_interface/H5N1_cumulative_table_archives/en/index.html). Due to such a high mortality rate, people can not help but worry that once the virus spreads in human, it will bring a fatal blow to human society.
Influenza virus is an enveloped, single stranded, negative-sense RNA virus that belongs to the genus influenza virus of the family Orthomyxoviridae. Its genome contains eight segments of RNA, and encode for more than 10 viral proteins. According to the difference in antigenicity and genetic characteristics of nucleoprotein (NP) and matrix protein (M), influenza virus can be classified into Influenza virus A, Influenza virus B, and Influenza virus C (Horimoto T. et al., Nat Rev Microbiol, 2005, 3(8): 591-600). Influenza A Virus (called Flu A for short) mutates fast, has a strong pathogenicity, and can cause worldwide pandemics. Influenza B Virus (called Flu B for short) mutates slow and can only cause small pandemics in local areas. Influenza C Virus (called Flu C for short) mutates slowest, has a weak pathogenicity, and can only infect pregnant women and children with a low resistance generally. In nature, there are a broad range of hosts for Flu A. Flu A can cause infection in animals such as human, horses and pigs, in addition to its natural host aquatic birds. Flu A has multiple subtypes which are different from each other greatly, and has become a research focus for influenza control and vaccine.
Flu A viruses are categorized into subtypes based on the antigenicity and genetic characteristics of the two surface antigens, i.e. hemagglutinin (HA) and neuraminidase (NA). There are 17 known HA subtypes (H1 to H17) and 10 known NA subtypes (N1 to N10) (Tong S. X. et al., PNAS. 2012, 109(11):4269-74). The Flu A subtypes epidemic in human beings include two HA subtypes (H1, H3) and two NA subtypes (N1, N2). Although highly pathogenic avian influenza virus H5N1 only infects human beings occasionally, it draws attention due to the high mortality rate.
The first line of defense to prevent influenza is neutralizing antibodies. The vaccine-induced neutralizing antibodies mainly target the membrane protein hemagglutinin (HA) on the surface of virus. HA protein on the surface of virus has a trimeric structure, and each HA monomer consists of HA1 domain and HA2 domain. HA1 forms a globule at the top of the trimer, contains receptor binding sites, and is a region essential for the virus to infect a host cell; HA1 also contains important antigenic sites, can induce generation of protective neutralizing antibodies in organisms, and thus is a key target for vaccine design (Wang T. T. et al., Nat Struct Mol Biol. 2009, 16: 233-234). HA2 is located at the base of the trimer, has a shape of stem, contains fusion peptide, and can mediate the fusion of viral envelope to host cell membrane; and some monoclonal antibodies against HA2 can inhibit the membrane fusion of influenza virus so as to achieve the effect of neutralizing virus (Wang T. T. et al., Nat Struct Mol Biol. 2009, 16: 233-234).
Influenza viruses have a high variability, particularly, HA mutates fastest. In current, traditional influenza vaccines are mainly directed to HA protein, and the influenza vaccines become ineffective easily because of virus antigenic drift caused by HA gene mutation. In order to keep influenza vaccines effective, WHO annually needs to monitor the mutation of the influenza virus strains epidemic last year, and select old influenza virus vaccines or establish new influenza virus vaccines as candidate influenza vaccines next year so as to retain the effective protection from epidemic influenza virus strains by inoculation of new vaccines every year. Therefore, development of “universal influenza vaccines” that are not affected by virus mutation becomes a research focus for development of new vaccines. The glycoprotein on the surface of influenza virus, i.e. “hemagglutinin (HA)”, is a main target for the development of universal influenza vaccines and immunotherapeutic medicines against influenza virus. The so-called “universal influenza vaccines” should comprise “highly conserved neutralizing epitopes” shared by different viral mutants, and can directly induce the generalization of “broad-spectrum neutralizing antibodies” to combat the infection by different virus mutants. Therefore, an important route for studying universal influenza vaccines and immunotherapeutic medicines against influenza is to look for highly conserved neutralizing epitopes on HA.
In addition, in a mouse animal model, a humanized HA monoclonal antibody specific to H1N1 and H5N1 has been demonstrated to be able to effectively treat experimental mice infected by influenza virus (Corti D. et al., J Clin Invest. 2010, 120:1663-1673). In clinic, polyclonal antibodies and monoclonal antibodies can be effectively applied to the prevention of infections by viruses such as Hepatitis A Virus, Hepatitis B Virus, rabies virus and respiratory syncytial virus (Sawyer L. A. et al., Antiviral Res. 2000, 47: 57-77). During Spanish influenza in 1918, it was reported that serum from human in convalescent stage was used for the treatment (Luke T. C. et al., Ann Intern Med. 2006, 145:599-609). The information suggests that antibodies can be used as alternative methods and tools for anti-viral therapy.
HA broad-spectrum monoclonal antibodies and highly conserved epitopes have been reported in many papers. Studies on broad-spectrum monoclonal antibodies and highly conserved epitopes against HA2 which is more conservative have become a major research focus (Throsby M. et al., PLoS One. 2009, 3: e3942; Sui J. et al., Nat Struct Mol Biol. 2009, 16: 265-273; Corti D. et al., J Clin Invest. 2010, 120: 1663-1673; Corti D. et al., Science. 2011, 333: 850-856). As early as 2009, Throsby et al. reported for the first time that a broad-spectrum neutralizing humanized monoclonal antibody CR6261 recognizing HA2 could neutralize all the influenza viruses belonging to Group 1 (including H1, H2, H5, H6, H8 and H9 subtypes) (Throsby M. et al., PLoS One. 2009, 3: e3942). In 2011, Corti D. et al. also obtained a humanized broad-spectrum neutralizing monoclonal antibody against HA2 by similar technology, which could neutralize 16 types of H subtype influenza viruses (Corti D. et al., Science. 2011, 333: 850-856), and became one of the most broad-spectrum neutralizing monoclonal antibodies against influenza reported recently. The discovery of these broad-spectrum monoclonal antibodies provides new hope for the development of broad-spectrum therapeutic monoclonal antibodies and universal influenza vaccines against influenza virus. The epitopes recognized by the monoclonal antibodies obtained in said studies are all located in a conservative region surrounding the fusion peptide on HA2, the main function of which is to mediate the membrane fusion of influenza virus, and the monoclonal antibodies do not recognize HA1 domain that have immune predominance. Therefore, it increases the uncertainty in the prospect of applying these antibodies and the recognized conservative epitopes to the treatment and prevention. Some studies show that the neutralizing monoclonal antibodies recognizing HA2 have a neutralizing activity reduced by 100-1000 folds for natural virus, as compared to pseudovirus, which may be due to the fact that the HA2 epitope of natural virus is not easily exposed and is difficult to be approached (Sui J. et al., Nat Struct Mol Biol. 2009, 16: 265-273; Corti D. et al., J Clin Invest. 2010, 120: 1663-1673).
As compared to HA2, HA1 of influenza virus forms a globule at the top of the trimer, contains a lot of neutralizing epitopes, and is easily accessible. Therefore, it is more possible to develop effective universal influenza vaccines and therapeutic antibodies by looking for highly conserved neutralizing epitopes on HA1. Since the subtypes of influenza virus are divided initially based on stereotyping of polyclonal antibodies and the pAb serum against one subtype has little cross-reactivity with virus of another subtype, it is generally believed that the neutralizing monoclonal antibodies specific to HA (particularly HA1) are serotype-specific and has no cross-reactivity with HA of another subtype. However, it is also found in some researches recently that the highly conserved neutralizing epitopes on HA1 can induce the generation of broad-spectrum monoclonal antibodies having cross-subtypes reactivity (Yoshida R. et al., PLoS Pathog. 2009, 5: e1000350; Krause J. C. et al., J Virol. 2011, 85: 10905-10908; Wrammert J. et al., J Exp Med. 2011, 208: 181-193). Since HA1 has a high variability, the reactive spectrum of the current HA1 monoclonal antibodies is narrow, as compared to that of HA2 monoclonal antibodies, and the HA1 monoclonal antibodies generally have a cross-neutralizing activity for 2-3 subtypes of influenza viruses. Yoshida et al. reported in 2008 that a HA1 broad-spectrum neutralizing monoclonal antibody, S139/1, could neutralize H1, H2, H3 and H13 subtypes of influenza viruses, but the neutralizing activity of the monoclonal antibody was only limited to a few virus strains for each of said subtypes (Yoshida R. et al., PLoS Pathog. 2009, 5: e1000350). After the 2009 pandemic H1N1 outbreak in 2009, many papers reported cross-neutralizing monoclonal antibodies against 2009 pandemic H1N1 and seasonal H1N1 influenza viruses, including the cross-neutralizing monoclonal antibody against 2009 pandemic H1N1 and H1N1 of 1918 (Krause J. C. et al., J Virol. 2010, 84: 3127-3130; Xu K. et al., Viral Immunol. 2011, 24: 45-56), and the cross-neutralizing monoclonal antibody against 2009 pandemic H1N1 and seasonal H1N1 epidemic recently (Krause J. C. et al., J Virol. 2011, 85: 10905-10908; Wrammert J. et al., J Exp Med. 2011, 208: 181-193). In addition, when screening a broad-spectrum neutralizing monoclonal antibody against HA2, Corti et al. also obtained a cross-neutralizing monoclonal antibody FE17 against HA1, which could cross-neutralize seasonal H1 and H5 subtypes of influenza viruses, and the epitope of which was located close to Ca2 antigenic region. These studies show that HA1 may contain conservative neutralizing epitopes, which are important for the research on universal influenza vaccines and immunotherapeutic medicines. Therefore, it is of importance and instructiveness for the development of broad-spectrum therapeutic antibodies and universal influenza vaccines against influenza, to look for broad-spectrum neutralizing monoclonal antibodies that recognize more highly conserved neutralizing epitopes on HA1 and to accurately localize the epitopes.